基于ITS序列和化学模式识别方法鉴定金蝉花及其混淆品独角龙

Identification of Cordyceps cicadae and Tolypocladium dujiaolongae based on ITS sequences and chemical pattern recognition method

基于ITS序列对金蝉花与独角龙进行分子鉴定,并建立HPLC指纹图谱结合化学模式识别方法,鉴别金蝉花及其混淆品独角龙。分别提取10批金蝉花与5批独角龙的基因组DNA,通过PCR扩增ITS序列并测序,利用MEGA 7.0软件比较二者的稳定差异位点并构建系统发育树;采用HPLC建立金蝉花与独角龙指纹图谱,运用相似度评价、聚类分析(CA)、主成分分析(PCA)和偏最小二乘-判别分析(PLS-DA)对其进行化学模式识别研究。结果表明金蝉花为Cordyceps cicadae,独角龙为Toly-pocladium dujiaolongae,二者的基原不同,且金蝉花与混淆品独角龙的ITS序列上共有115个稳定差异位点,构建的系统发育树可以有效的区分二者;建立了18批金蝉花与5批独角龙的HPLC指纹图谱,CA、PCA和PLS-DA结果一致,均能很好地区分二者,表明金蝉花与独角龙在化学成分上存在较大的差异,且PLS-DA结果显示尿苷、鸟苷、腺苷和N^(6)-(2-羟乙基)腺苷等6个成分是导致二者差异的主要标志性成分。ITS序列和HPLC指纹图谱结合化学模式识别方法均可以作为金蝉花与独角龙的鉴定和鉴别手段。

Based on ITS sequences,the molecular identification of Cordyceps cicadae and Tolypocladium dujiaolongae was carried out,and high-performance liquid chromatography(HPLC)fingerprint combined with chemical pattern recognition method was established to differentiate C.cicadae from its adulterant T.dujiaolongae.The genomic DNA from 10 batches of C.cicadae and five batches of T.dujiaolongae was extracted,and ITS sequences were amplified by PCR and sequenced.The stable differential sites of these two species were compared and the phylogenetic tree was constructed via MEGA 7.0.HPLC was used to establish the fingerprints of C.cicadae and T.dujiaolongae,and similarity evaluation,cluster analysis(CA),principal component analysis(PCA),and partial least squares discriminant analysis(PLS-DA)were applied to investigate the chemical pattern recognition.The result showed that the sources of these two species were different,and there were 115 stable differential sites in ITS sequences of C.cicadae and T.dujiao-longae.The phylogenetic tree could distinguish them effectively.HPLC fingerprints of 18 batches of C.cicadae and 5 batches of T.dujiaolongae were established.The results of CA,PCA,and PLS-DA were consistent,which could distinguish them well,indicating that there were great differences in chemical components between C.cicadae and T.dujiaolongae.The results of PLS-DA showed that six components such as uridine,guanosine,adenosine,and N^(6)-(2-hydroxyethyl)adenosine were the main differential markers of the two species.ITS sequences and HPLC fingerprint combined with the chemical pattern recognition method can serve as the identification and differentiation methods for C.cicadae and T.dujiaolongae.